A protein similar to alpha-actinin has been isolated from unfertilized sea urchin eggs. This protein co-precipitated with actin from an egg extract as actin bundles. Its apparent molecular weight was estimated to be ~95,000 on an SDS gel: it co-migrated with skeletalmuscle alpha-actinin. This protein also co-eluted with skeletal muscle alpha-actinin from a gel filtration column giving a Stokes radius of 7.7 nm, and its amino acid composition was very similar to that of alpha-actinins. It reacted weakly but significantly with antibodies against chicken skeletal muscle alpha-actinin. We designated this protein as sea urchin egg alphaactinin. The appearance of sea urchin egg a/pha-actinin as reveaFed by erectron microscopy using the low-angle rotary shadowing technique was also similar to that of skeletal muscle alpha-actinin. -I-his protein was able to cross-link actin filaments side by side to form large bundles. The action of sea urchin egg alpha actinin on the actin filaments was studied by viscometry at a low-shear rate. It gelled the F-actin solution at a molar ratio to actin of more than 1:20, at pH 6-7.5, and at Ca ion concentration <1 #M. The effect was abolished by the presence of tropomyosin. Distribution of this protein in the egg during fertilization and cleavage was investigated by means of microinjection of the rhodamine-labeled protein in the living eggs. This protein showed a uniform distribution in the cytoplasm in the unfertilized eggs. Upon fertilization, however, it was concentrated in the cell cortex, including the fertilization cone. At cleavage, it seemed to be concentrated in the cleavage furrow region.Actin is a protein that forms filamentous polymers at physiological salt concentrations. In nonmuscle cells, motility and maintenance of cell shape are largely dependent on actinbased structures. These structures show a variety of morphologies: some are ordered arrays of actin filaments and others are random networks. These three-dimensional structures are considered to be constructed of actin plus actin cross-linking proteins since purified actin does not form such structures in vitro under physiological salt conditions. One of these proteins is alpha-actinin. This protein was discovered by Ebashi and colleagues (8, 32) in rabbit skeletal muscle and was localized to the Z disk of the sarcomere structure (33). In nonmuscle cells, its presence in the stress fibers of cultured mammalian cells was strongly suggested by an immunofluorescence study using antibodies against skeletal muscle alph-actinin (23). Since then, proteins that are similar to skeletal or smooth muscle alpha-actinin have been isolated from various types of mammalian tissues and cells: brain (42), Ehrlich tumor cells ("actinogelin," reference 35), HeLa cells (5), platelets (40), chromaffin granules (1), kidney (20), and Sarcoma 180 ascites (53). Such proteins have also been isolated from primitive organisms such as Acanthamoeba (38) and Dictyostelium (6,10). These proteins may be important in the organization of aclin ...

Porcine brain tubulin labeled with fluorescein isothiocyanate (FITC) was able to polymerize by itself and co-polymerize with tubulin purified from starfish sperm flagella. When we injected the FITC-labeled tubulin into unfertilized eggs of the sand dollar, Clypeaster japonicus, and the eggs were then fertilized, the labeled tubulin was incorporated into the sperm aster. When injected into fertilized eggs at streak stage, the tubulin was quickly incorporated into each central region of growing asters. It was clearly visualized that the labeled tubulin, upon reaching metaphase, accumulated in the mitotic apparatus and later disappeared over the cytoplasm during interphase. The accumulation of the fluorescence in the mitotic apparatus was observed repeatedly at successive cleavage. After lysis of the fertilized eggs with a microtubule-stabilizing solution, fluorescent fibrous structures around the nucleus and those of the sperm aster and the mitotic apparatus were preserved and coincided with the fibrous structures observed by polarization and differential interference microscopy. We found the FITC-labeled tubulin to be incorporated into the entire mitotic apparatus within 20-30 s when injected into the eggs at metaphase or anaphase. This rapid incorporation of the labeled tubulin into the mitotic apparatus suggests that the equilibrium between mitotic microtubules and tubulin is attained very rapidly in the living eggs. Axonemal tubulin purified from starfish sperm flagella and labeled with FITC was also incorporated into microtubular structures in the same fashion as the FITC-labeled brain tubulin. These results suggest that even FITC-labeled heterogeneous tubulins undergo spatial and stage-specific regulation of assembly-disassembly in the same manner as does sand dollar egg tubulin.

Actin from sea urchin eggs was fluorescently labeled with fluorescein isothiocyanate (FITC), N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM), or 5-iodoacetamidofluorescein (IAF) and microinjected into sea urchin eggs and oocytes. It distributed evenly in the cytoplasm of unfertilized eggs. Upon fertilization, actin accumulated first around the sperm binding site and, soon afterwards, in the fertilization cone. The accumulation propagated all over the cortex after a latent period of 10-20 sec. In the case of Clypeuster juponicus eggs, propagation of the accumulation coincided with a shape change in the egg, suggesting that the accumulated actin in the cortex generates forces. FITC-actin was incorporated into microvilli and retained in the cortex after cleavage. On the other hand, DACM-or IAF-actin was not incorporated into microvilli and was dispersed from the cortex by cleavage. These differences may be attributable to differences in the properties of the actins labeled at different sites. After photobleaching by laser light irradiation, FITC-or IAF-actin redistributed in the cortex of fertilized egg as quickly as it did before fertilization. When an unfertilized egg was injected with both actin and a calcium buffer (intracellular free Ca2+ concentration 9 pM), the actin accumulation was similar to that during fertilization but without the latent period. This suggests that the accumulation depended on the increase in the intracelluiar free Ca2' concentration. When the unfertilized egg was injected with 0.2 M EGTA after injection of labeled actin and then inseminated, it accumulated only in the protrusion of cytoplasm where the sperm had entered, and fertilization was not completed. In immature oocytes, the accumulation was observed in the cortical region, including the huge protrusion of the cytoplasm where the sperm had entered. These results suggest that actin accumulation in the sperm binding site plays an important role in the sperm reception mechanism of the egg.

No abstract

No abstract